Single-Molecule Imaging of EGFR Signalling on the Surface of Living Cells

articles Single-molecule imaging of EGFR signalling on the surface of living cells Yasushi Sako*, Shigeru Minoguchi* and Toshio Yanagida*†‡ *Department of Physiology and Biosignalling, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan †Single Molecule Processes Project, ICORP, JST, 2-4-14 Senba-Higashi, Mino 562-0035, Japan ‡e-mail: [email protected] The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single- molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF–EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization. ingle-molecule imaging is an ideal technology by which to Results study the molecular mechanisms of biological reactions in Visualization of single molecules on the surface of living cells. As Svitro1–3. Here we extend this technology to real-time obser- a probe for detection of single molecules in living cells, we used vations of the binding of fluorescent-dye-labelled EGF to its a fluorescent dye, Cy3, conjugated to mouse EGF. As mouse EGF receptor (EGFR), dimerization of EGF–EGFR complexes, and has only one reactive amino residue (at the amino terminus), it autophosphorylation of EGFR in the plasma membrane. can be labelled with amino-reactive Cy3 dye with a dye:protein Although dimerization and autophosphorylation are known to ratio of exactly 1:1. be essential for signal transduction of EGFR4,5, the mechanisms Under an objective-type total internal reflection (TIR) fluores- underlying these events have not been fully understood until cence microcope3, Cy3-labelled EGF (Cy3–EGF) was added to the now. culture medium of A431 cells (Fig. 1). The background autofluores- a Evanescent field c Focus Apical Basal Medium Coverslip Oil θ Objective TIR (apical) Laser 1 µm b Apical imaging 10 µm Basal imaging TIR (basal) Illumination θ1 Laser θ2 θ θ Epi 1> 2 Figure 1 TIR microscopy for visualization of single molecules on the cell on both apical and basal surfaces. In this case, spots on the basal surface would be surface. a, TIR microscopy. An objective-type TIR microscope was used. b, For excited by epifluoresence illumination. Because of excitation of Cy3–EGF in the imaging of the basal cell surface, a laser beam was reflected between a coverslip basolateral surface and free Cy3–EGF in the evanescent field, background and the culture medium. For imaging of the apical plasma membrane, the incident fluorescence was higher on the apical surface than on the basal surface. Even in this θ θ ) angle of the laser beam was reduced ( 1 > 2 and the laser beam was reflected condition, individual Cy3–EGF molecules could be visualized on the apical surface between the cytoplasm and culture medium. c, To demonstrate the effect of the (inset). Under basal TIR mode, spots were only observed on the basal surface illumination mode, images of A431 cells were taken in the same field under different because of the limited excitation depth of TIR. Under the epifluorescence illumination modes in the presence of 100 ng ml–1 Cy3–EGF. The cells were 2–3 µm configuration, background from fluorescent EGF in the culture medium was high and thick, and so, by changing the focus, we could easily distinguish between the apical no fluorescent spots on the cell surface were detected. surface and the basal surface. Under apical TIR mode, fluorescent spots were shown 168 © 2000 Macmillan Magazines LtdNATURE CELL BIOLOGY | VOL 2 | MARCH 2000 | cellbio.nature.com articles acb d Cy3-EGF e 800 25 400 20 100µW µm –2 0 15 800 τ=2.5s 10 400 5 0 0 800 0 4 8 1216 20 400 15 0 10 τ=6.8s 40µW µm –2 800 5 Frequency (%) Frequency 400 0 0 0 4 8 1216 20 15 Fluorescence intensity (AU) Cy5-EGF 800 10 τ=13.9s 20µW µm–2 400 5 0 0 0 246810 0 4 8 1216 20 Time (s) Duration (s) Figure 2 Visualization of Cy3–EGF on the plasma membrane of living A431 (30 frames every second) and plotted against time. An HeNe laser (632.8 nm) was cells. a, b, Cy3–EGF at a final concentration of 0.2 ng ml–1 was added to the used for observation of Cy5–EGF. AU, arbitrary unit. e, Histograms of the time culture medium of living A431 cells under a TIR fluorescence microscope. Images difference between the appearance and disappearance of Cy3–EGF spots under were acquired immediately (a) and 1 min (b) after the addition of Cy3–EGF. c, A lasers of variable excitation powers. The lines represent the fitting of the results mixture of Cy3–EGF (0.2 ng ml–1) and EGF (200 ng ml–1) was added to the culture to a single exponential function. τ indicates the time constant of the fit function. medium of A431 cells. This image was acquired 1 min after the addition of Cy3– Multiple correlation coefficients (R) of the fitting are 0.991 (100 µW µm–2), 0.916 EGF and EGF. Scale bar represents 10 µm. d, The change in fluorescence intensity (40 µW µm–2) and 0.741 (20 µW µm–2). of individual Cy3 (or Cy5)–EGF spots on living cells was measured frame by frame cence of cells before addition of Cy3–EGF was low, except at large lifetime is proportional to the excitation laser power (Fig. 2e). This vesicles in the perinuclear region. Soon after the addition of Cy3– relationship between the lifetime and excitation power indicates EGF, fluorescent spots appeared on the cell surface; the density of that the duration of fluorescence was determined by the rate of pho- these spots increased with time (Fig. 2a, b). Cy3–EGF was not tobleaching of Cy3–EGF. Thus it is likely that the sudden disappear- detectable as fluorescent spots in the culture medium because of ance of the Cy3–EGF spots was caused by photobleaching, not by brownian movement in solution. In addition, the background flu- dissociation from EGFR or by endocytosis into the cytoplasm. Sin- orescence caused by Cy3–EGF in the medium was low because of gle-step photobleaching is strong evidence that the fluorescent the low excitation depth of TIR microscopy. Binding of Cy3–EGF spots represented single Cy3–EGF molecules on the cell surface1. to the cell surface was completely inhibited by addition of excess We conjugated a monoclonal anti-EGFR antibody (EGFR1) unlabelled EGF at the same time that Cy3–EGF was added (Fig. 2c), with Cy3 (generating Cy3–EGFR1), and allowed this to bind to indicating specific binding of Cy3–EGF to the cell-surface EGFR. A431 cells in the same way as Cy3–EGF. The concentration of Cy3– Cy3–EGF did not bind to CHO-K1 cells, which lack EGFR6 (data EGFR1 was low (0.1 µg ml–1) so as not to induce aggregation of not shown). EGFR. As EGFR1 can be labelled with multiple Cy3 molecules, the By carefully adjusting the incident angle of the excitation laser intensity distribution of Cy3–EGFR1 spots on the cell surface was beam, we were able to visualize Cy3–EGF on both apical and baso- fitted to a sum of two or three Gaussian distribution functions (Fig. lateral plasma membranes by TIR microscopy (Fig. 1b, c). A slight 3b, c). The results of this fitting are consistent with Poisson distri- difference in the refractive indices of the cytoplasm and the culture butions of the number of Cy3 molecules to EGFR1 molecules, indi- medium resulted in imaging of the apical surface of the cell mem- cating that the first, the second and the third components represent brane. Here we studied mainly the apical surface (see Supplemen- EGFR1 conjugated with one, two or three Cy3 molecules, respec- tary Information). tively. We followed the change in fluorescence intensity of each Cy3– As, in the intensity distribution of Cy3–EGF (Fig. 3a), the mean EGF spot in living A431 cells (Fig. 2d). In many cases, a spot of the first component was the same as those for Cy3–EGFR1, we appeared suddenly on the cell surface, emitted almost constant flu- conclude that this component contains single Cy3–EGF molecules. orescence, then disappeared suddenly. Histograms of the time dif- Binding of EGF to EGFR induces dimerization of EGFR4, which is ference between the appearance and the disappearance of each spot indispensable for EGF signalling. The second component, which were well fit with a single exponential function. The inverse of the emits a signal twice as intense as the first component, may represent NATURE CELL BIOLOGY | VOL 2 | MARCH 2000 | cellbio.nature.com © 2000 Macmillan Magazines Ltd 169 articles 86 a + Cy3-EGF a 16 4 3 Cy3-EGF 12 2 n=195 intensity 1 8 012 34 14 Relative fluorescence Relative Time (min) 4 b 16 0 0 500 1,000 1,500 54 0-20s b 16 12 83 (81) 46 n=141 Cy3-EGFR1 8 12 D/P=0.4 4 8 n=200 0 17 (16) 0 100 200 300 400 500 4 Frequency (%) Frequency 16 37 0 63 0 500 1,000 1,500 12 20-40s c 16 n=250 63 (60) 8 Cy3-EGFR1 12 D/P=1.0 4 n=192 8 (%) Frequency 0 35 (30) 0 100 200 300 400 500 4 2 (10) 16 31 0 12 40-60s 0 500 1,000 1,500 69 n=234 Fluorescence intensity (AU) 8 Figure 3 Distribution of the fluorescence intensity of Cy3–EGF and Cy3– 4 EGFR1 1 on the surface of living cells.

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